Yaohui Zhu

Nerve growth factor modulates TRPV1 expression and function and mediates pain in chronic pancreatitis.

BACKGROUND & AIMS The pathogenesis of pain in chronic pancreatitis (CP) is poorly understood and treatment remains difficult. We hypothesized that nerve growth factor (NGF) plays a key role in this process via its effects on the transient receptor potential vanilloid 1, TRPV1. METHODS CP was induced by intraductal injection of trinitrobenzene sulfonic acid in rats. After 3 weeks, anti-NGF antibody or control serum was administered daily for 1 week. Pancreatic hyperalgesia was assessed by nocifensive behavioral response to electrical stimulation of the pancreas as well as by referred somatic pain assessed by von Frey filament testing. TRPV1 currents in pancreatic sensory neurons were examined by patch-clamp. The expression and function of TRPV1 in pancreas-specific nociceptors was examined by immunostaining and quantification of messenger RNA levels. RESULTS Blockade of NGF significantly attenuated pancreatic hyperalgesia and referred somatic pain compared with controls. It also decreased TRPV1 current density and open probability and reduced the proportion of pancreatic sensory neurons that expressed TRPV1 as well as levels of TRPV1 in these neurons. CONCLUSIONS These findings emphasize a key role for NGF in pancreatic pain and highlight the role it plays in the modulation of TRPV1 expression and activity in CP.

Systemic administration of anti-NGF increases A-type potassium currents and decreases pancreatic nociceptor excitability in a rat model of chronic pancreatitis.

We have previously shown that pancreatic sensory neurons in rats with chronic pancreatitis (CP) display increased excitability associated with a decrease in transient inactivating potassium currents (I(A)), thus accounting in part for the hyperalgesia associated with this condition. Because of its well known role in somatic hyperalgesia, we hypothesized a role for the nerve growth factor (NGF) in driving these changes. CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in rats. After 3 wk, anti-NGF antibody or control serum was injected intra-peritoneally daily for 1 wk. This protocol was repeated in another set of experiments in control rats (receiving intraductal PBS instead of TNBS). Pancreatic nociceptors labeled with the dye Dil were identified, and patch-clamp recordings were made from acutely dissociated DRG neurons. Sensory neurons from anti-NGF-treated rats displayed a lower resting membrane potential, increased rheobase, decreased burst discharges in response to stimulatory current, and decreased input resistance compared with those treated with control serum. Under voltage-clamp condition, neuronal I(A) density was increased in anti-NGF-treated rats compared with rats treated with control serum. However, anti-NGF treatment had no effect on electrophysiological parameters in neurons from control rats. The expression of Kv-associated channel or ancillary genes Kv1.4, 4.1, 4.2, 4.3, and DPP6, DPP10, and KCHIPs 1-4 in pancreas-specific nociceptors was examined by laser-capture microdissection and real-time PCR quantification of mRNA levels. No significant differences were seen among those. These findings emphasize a key role for NGF in maintaining neuronal excitability in CP specifically via downregulation of I(A) by as yet unknown mechanisms.

Anatomical and functional characterization of a duodeno-pancreatic neural reflex that can induce acute pancreatitis

Neural cross talk between visceral organs may play a role in mediating inflammation and pain remote from the site of the insult. We hypothesized such a cross talk exists between the duodenum and pancreas, and further it induces pancreatitis in response to intraduodenal toxins. A dichotomous spinal innervation serving both the duodenum and pancreas was examined, and splanchnic nerve responses to mechanical stimulation of these organs were detected. This pathway was then excited on the duodenal side by exposure to ethanol followed by luminal mustard oil to activate transient receptor potential subfamily A, member 1 (TRPA1). Ninety minutes later, pancreatic inflammation was examined. Ablation of duodenal afferents by resiniferatoxin (RTX) or blocking TRPA1 by Chembridge (CHEM)-5861528 was used to further investigate the duodeno-pancreatic neural reflex via TRPA1. ~40% of dorsal root ganglia (DRG) from the spinal cord originated from both duodenum and pancreas via dichotomous peripheral branches; ~50% splanchnic nerve single units responded to mechanical stimulation of both organs. Ethanol sensitized TRPA1 currents in cultured DRG neurons. Pancreatic edema and myeloperoxidase activity significantly increased after intraduodenal ethanol followed by mustard oil (but not capsaicin) but significantly decreased after ablation of duodenal afferents by using RTX or blocking TRPA1 by CHEM-5861528. We found the existence of a neural cross talk between the duodenum and pancreas that can promote acute pancreatitis in response to intraduodenal chemicals. It also proves a previously unexamined mechanism by which alcohol can induce pancreatitis, which is novel both in terms of the site (duodenum), process (neurogenic), and receptor (TRPA1).

740 The Role of SMAD Signaling in the Pathogenesis of Pain in Chronic Pancreatitis

Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cytokine mainly produced by T cells, beside B cells and mast cells. Mounting evidence suggests that there may be a specialized subset of T cells dedicated to produce IL-9 which are so called Th9 cells. In these cells the IL-9 gene is regulated by transcription factors like PU.1 and IRF4. Also many cytokines have been investigated so far in experimental models of chronic intestinal inflammation, the role of IL-9 is largely unidentified. But high concentrations of IL-9 in the colon tissue during colitis reveal the important role of this cytokine. Methods: For the analysis of IL-9 in the development of chronic intestinal inflammation IL-9 deficient mice were used in oxazolone-colitis model. Miniendoscopic analysis has been done to monitor the manifestation of the colitis. The inflamed colon was isolated and histological sections were taken for immunohistofluorescent staining and real-time PCR analysis. For therapeutically treatment wildtype mice were given 40μg of specific anti IL-9 antibody to prevent the emergence of colitis. Results: In the experimental oxazolone-colitis model the IL-9KO mice were protected. This became evident in the miniendoscopic analysis and in the HE staining. Immunofluorescence staining shows a decrease of the IL-9 regulating transcription factor PU.1 in the IL-9 deficient mice, indicating the involvement of PU.1 in the IL-9 production. This is consistent with the fact that PU.1 is higher expressed in human biopsies of colitis patients, which illustrates the pro-inflammatory role of IL-9 in patients suffering from inflammatory bowel disease. Double staining of CD4+ and PU.1 and EPCAM and PU.1 respectively suggests that there are specialized T cells expressing the transcription factor PU.1 implying a role of Th9 cells in colitis. Further analysis of the pro-inflammatory effect of IL-9 showed that the blockage of high IL-9 concentrations with a specific anti IL-9 antibody in wildtype mice lead to a protection in oxazolone-colitis model. Conclusion: Here, we have identified a central pathogenic role for Th9 cells in chronic intestinal inflammation. This is based on the fact that IL-9 is increased in inflamed colon tissue and IL-9KO mice are protected in the experimental oxazolone-colitis model. Furthermore, administration of a blocking anti IL-9 antibody before the manifestation of colitis has a protective effect. Thus IL-9 emerges as a potentially new therapeutic target for inflammatory bowel diseases.